中国兽医科学2012，Vol.42Issue(2)：176-181,6.
狂犬病“靶向性”Th表位DNA疫苗的构建与瞬时表达研究
Construction and transient expression study of target DNA vaccine expressing Th antigenic epitopes of rabies virus
摘要
Abstract
The BALB/c mouse invariant chain P31 was obtained by using RT-PCR and a P31 mutant was obtained by site-directed mutagenesis to the Nco Ⅰ restriction endonuclease site at 461 nt with the amino acid coding sequence no changes. Then the CLIP domain was substituted by the Th antigenic epitopes of rabies virus nucleoprotein,glycoprotein and NS protein. PCR was performed to obtain the P31-Th sequence that containing the Kozak regulative sequence to increase the expression efficiency of target genes.The P31-Th genes were then cloned into the multiple cloning sites of the eukaryotic expression vector pCI-neo and the recombinant plasmids were confirmed by PCR and restriction enzyme digestion methods.Then the Th antigenic epitopes expressing plasmids were transfected respectively into COS-7 cells. Western-blotting was used to detect transient expression products of target genes. Sequencing analysis showed that the BALB/c mouse P31 gene CDS was 648bp in length encoding 215 amino acids and there were multiple different nucleotides or amino acid sites compared with the sequence in the GenBank（NM_010545）.The site-mutant P31 was obtained after site-directed mutagenesis,and the P31-Th expressing plasmids were successfully constructed. The transiently expression products of target genes in COS-7 cells could react with the rabbit anti-P31 polyclonal antibodies.These results indicated that the target DNA vaccines expressing Th antigenic epitopes of rabies virus was successfully constructed and could be used for further immune experiments in mice.关键词
狂犬病病毒Th表位/BALB/c小鼠恒定链/定点突变/定向克隆/瞬时表达Key words
Th antigenic epitopes of rabies virus/BALB/c mouse invariant chain/site-directed mutagenesis/transient expression/directed cloning分类
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肖跃强,管宇,谢金文,郭广君,魏风,刘吉山,沈志强..狂犬病“靶向性”Th表位DNA疫苗的构建与瞬时表达研究[J].中国兽医科学,2012,42(2):176-181,6.基金项目
山东省青年自然科学基金项目 （）
